We are currently exploring the mechanisms by which chaperones and proteases act together to regulate protein activity and degradation. More specifically we are investigating the role of E. coli ClpA, an ATP-dependent molecular chaperone and the regulatory component of the ATP-dependent ClpAP protease, in protein remodeling and degradation. We discovered that ClpA converts plasmid P1 RepA dimers into monomers, thereby activating the latent DNA binding activity of RepA and that ClpA in conjunction with ClpP degrades RepA.One aspect of the current model of the interplay of proteases and chaperones in protein quality control is that the ATPase components of ATP-dependent proteases regulate proteolysis by reactivating less severely damaged proteins and translocating irreversibly damaged proteins to the proteolytic component. A prediction of this model is that ClpA, while in a complex with ClpP, can carry out both chaperone activity and proteolysis. To test this, we simultaneously monitored chaperone activity and protease activity during a single cycle of RepA binding to ClpAP and ATP- dependent release. We demonstrated that chaperone and protease activities occur concurrently in ClpAP complexes within a single cycle of binding and release.This result was substantiated with studies of ClpA(K220V), a mutant with valine substituted for lysine in the first ATP binding site. We found that the mutant protein was unable to carry out molecular chaperone activity in the RepA activation reaction. Surprisingly, we observed that ClpA(K220V) was able to degrade RepA in combination with ClpP. This result appeared to contradict our working model that the chaperone activity functions early in the pathway of degradation by unfolding substrates to physically allow their passage into the proteolytic chamber of ClpP. Importantly, we found that ClpP not only facilitates degradation of RepA, it also restores the ability of ClpA(K220V) to activate RepA. Chemically inactivated ClpP also restored the chaperone activity of ClpA(K220V), in this case in the absence of degradation. ClpP must remain bound to ClpA(K220V) for this effect. These results demonstrate that both chaperone and proteolytic activities of the mutant complex occur concurrently.Our results point out an important new element in the model of energy-dependent proteolysis. They suggest that substrates bound to the complex of the proteolytic and ATPase component can be partitioned between release/reactivation and translocation/degradation. Thus, the fate of a substrate to be degraded instead of refolded is not simply a consequence of a proteolytic component associating with an ATPase component. - DNA binding proteins, DNA replication, heat-shock proteins, protein folding, protein structure, molecular chaperones, - Neither Human Subjects nor Human Tissues